Progressive Multifocal Leukoencephalopathy (PML), a demyelinating encephalopathy caused by the human JC polyomavirus, is a life-threatening complication of natalizumab therapy for relapsing multiple sclerosis. Seropositivity for JCV, prior immunosuppression, and > 24 month of natalizumab treatment are the only identified factors known to increase risk for PML, but their utility is limited because of the high prevalence of JCV infection and the heterogeneity of immunosuppressive regimens patients receive before starting natalizumab. No anti-JCV agents are available. Elucidation of the pathogenesis of PML and immunosurveillance mechanisms that keep JCV replication in check are required to adequately assess risk for PML in MS individuals receiving natalizumab. Polyomaviruses are highly species specific, with humans being the only host reservoir for JCV. A tractable animal model is urgently needed to understand the immunologic and virologic determinants of JCV-induced PML, and to provide a preclinical platform to assess the in vivo efficacy of novel anti-JCV compounds. Using mouse polyomavirus (MPyV), we have developed a mouse model of polyomavirus-induced CNS disease. We propose applying this MPyV-CNS infection model to address two Aims based on the following hypotheses: (1) by blocking T cell extravasation across the blood-brain barrier into the CNS parenchyma, natalizumab prevents maintenance of brain-resident virus-specific CD8 T cells required for antiviral immunosurveillance; and (2) because JCV in PML patients harbor novel mutations in their capsid protein, the dominant target for humoral-mediated immune defense, virus-neutralizing antibodies select viral variants that acquire neurotropism. For Aim 1, we have constructed MHC class I and class II tetramers to visualize MPyV-specific CD8 and CD4 T cells by flow cytometry, and have developed an MPyV- specific TCR transgenic mouse to monitor the fate of anti-MPyV CD8 T cells in the CNS. For Aim 2, we will create a library of capsid mutant MPyVs by PCR-based random mutagenesis, and by combining iterative virus passaging in vivo with next-generation sequencing, determine whether neutralizing MPyV antibodies select mutations that confer neurovirulence. We will also attempt to create a hybrid virus expressing the capsid proteins of JCV (to confer JCV host cell specificity) and having the noncoding control elements, origin of replication, and nonstructural proteins of MPyV (to enable replication in the mouse). This novel hybrid virus will allow us to determine the functional significance of the capsid mutations in JCV-PML variant viruses.